PROCESS TRANSFER TO PILOT-SCALE FERMENTATION SYSTEM
The cGMP pilot plant fermentation system had a 100 L maximum working volume and had previously been used as a seed tank to
support larger scale mammalian and microbial cultivation. Thus, it was expected that this system would have certain operating
constraints that might require further adjustment of the laboratory-scale process prior to implementation. To prepare for
the transfer, the pilot plant bioreactor was characterized and compared to the laboratory system.
The production of a high-density cGMP cell bank was initiated prior to the planned cGMP fermentation. The protocol used for
creation of the cGMP cell bank was similar to that used in the laboratory. However, additional testing of the cell bank for
viability, purity (bacterial and fungal contaminants), host strain identity, and absence of bacteriophage was undertaken to
ensure compliance with existing guidelines.
Figure 7: Characterization strategy for fermentation equipment.
Equipment-related parameters for characterization were selected based on literature review, past experience with E. coli fermentation, and prior knowledge of the equipment (see Figure 7) (10–16). These included certain theoretical characteristics,
as well as experimentally measured characteristics of the fermentation vessels.
Table II: Fermentation vessel specifications.
Characterization of the fermentation equipment was initiated with a review of system specifications (see Table II). Theoretical
characteristics of the fermentation systems selected to serve as comparative metrics were geometric similarity, maximum impeller
tip speed, maximum power input per unit volume, and superficial velocity at 1 vvm (see Table III) (12, 15, 17, 18). The difference
in the geometric similarity parameter for the 100-L vessel and 15-L vessel reflected the difference in aspect ratio of the
two tanks. Calculations for impeller tip speed indicated that higher tip speeds were achieved at the 100-L scale due to larger
impeller size. To estimate power input per unit volume, a range from 5.2 to 6.5 was assumed for the power number (NP) based on the literature, and both values were used for calculations (17). Again, it was observed that for a given agitation
rate, the power input per unit volume was higher for the 100-L fermenter, due to a larger impeller size. The superficial velocity
was much lower for the 100-L vessel compared to the 15-L vessel for the same gas flow rate expressed in vessel volume per
Table III: Summary of characterization results.
Liquid level with respect to internal features was measured with and without agitation or sparging for the two fermentation
vessels. This was intended to help determine a starting batch volume that ensured complete immersion of impellers and hence
optimum oxygen transfer (see Figure 8).
Figure 8: Bioreactor system characterization. (A) Liquid level with respect to internal features for the 15-L vessel. (B)
Liquid level with respect to internal features for the 100-L vessel.
The liquid phase volumetric oxygen mass transfer coefficient (kLa h-1), was experimentally measured to provide a quantitative estimate of the oxygen transfer capability of the system. Since agitation
rate and airflow rate are the two most common means of controlling oxygen transfer and hence DO, it was decided to estimate
kLa under different combinations of agitation and airflow rates, while keeping all other parameters the same. In order to maintain
simplicity and transferability of the test protocol, the static gassing-out method was selected to estimate kLa, with water as the test medium (13, 19, 20). This test involved filling a vessel with water and alternately sparging with
nitrogen or air to deoxygenate and re-oxygenate the test medium, respectively. The rate of oxygen re-absorption was recorded
with an oxygen electrode and provided an estimate of the kLa through the equation:
where, C is concentration of oxygen in the medium at time 't' (mM/L), t is time in h, kLa is liquid phase mass transfer coefficient (h-1 ), and C* is saturation/equilibrium oxygen concentration in medium under experimental conditions (mM/L).
At the 15-L scale, the experimental conditions selected were between 500–1200 rpm for agitation and 10–15 Lpm for airflow.
At the 100-L scale, the experimental conditions selected were between 150–600 rpm for agitation and 60–90 Lpm for airflow.
The temperature and pressure were set to operating values for the Q-beta VLP fermentation process. 11 L and 59 L of water
were used in the laboratory vessel and pilot plant vessel respectively. Minitab 15 from Minitab Inc. (State College, PA) was
used to select the experimental points at each scale and for subsequent analysis of the results. The kLa data at each scale was used to create a response surface with respect to agitation and airflow by employing a full quadratic
model. Minitab was then able to calculate the optimum kLa for each response surface, which provided an estimate of the maximum kLa that could be achieved at a particular scale. The response surface model quality was analyzed by examining the normal plot
of residuals, as well as the model fit for each point. Maximum kLa values obtained were 137 h-1 for the 15-L scale and 118 h-1 for the 100-L scale (see Figure 9, Table III). A lower kLa at the 100-L scale was not unexpected, as the pilot bioreactor was primarily designed as seed culture vessel rather than
a high-density production culture vessel. A quadratic model was used for data interpretation because this was a convenient
built in feature provided by Minitab. Other models would also be expected to give similar results, such as the commonly used
correlation represented by kLa = k(P0/V)α (Us)β , where k, α and β are empirical constants.
Figure 9: (A) Bioreactor system characterization – determination of kLa in water for the 15-L scale lab vessel. Agitation and airflow were varied, temperature and pressure were set at 0.5 Bar
and 30 °C respectively. The vessel contained 11 L of water, completely submerging two impellers. (B) Bioreactor system characterization
– determination of kLa in water for the 100-L pilot scale vessel. Agitation and airflow were varied, temperature and pressure were set at 0.5 Bar
and 30 °C respectively. The vessel contained 59 L of water, completely submerging two impellers.
Heat transfer and heat load
To estimate and compare the cooling capacity of the fermentation systems at 30 °C, each vessel was filled with water, and
the temperature set-point was repeatedly switched between 40 °C and 20 °C. All other parameters were set to operating values
for the Q-beta VLP fermentation process. The recorded temperature profiles allowed calculation of cooling capacity, assuming
the specific heat capacity of water at 30 °C as 1 kCal/kg –°C. Cooling capacity of the 15-L fermentation system was estimated
at 21 kCal/min using 10 L water with 10 Lpm air flow, 755 rpm agitation at 30 °C (108 kCal/min–m2 of heat transfer area) (see Table II). The cooling capacity of the 100-L fermentation system was estimated at 60 kCal/min
using 59 L water with 59 Lpm air flow, and 415 rpm agitation at 30 °C (90 kCal/min–m2 of heat transfer area).
The theoretical heat load was estimated based on OUR (9, 14, 21). For a rapidly growing culture using glycerol, assuming complete
oxidation of the carbon source, and that all oxygen transferred into the liquid phase is used for the reaction:
where QH is the metabolic heat released in kCal/L–h, OUR is expressed as mM/L–h, and ΔHGlycerol is heat of formation of glycerol (0.397 kCal/mM). The heat load corresponding to an OUR of 250 mM/L–h was estimated at 5
kCal/min for an 11-L batch and 28 kCal/min for a 59-L batch.
Estimating water loss was considered important because the 100-L vessel did not include an exhaust condenser. Maximum theoretical
water loss was estimated using a mass balance, assuming air entering the vessel was bone dry and air exiting the vessel was
fully saturated with water vapor. Calculations assumed 59 Lpm air flow at 30 °C and atmospheric pressure. The water loss was
also experimentally determined after holding water in the vessel under operating conditions for 16 h (59 L water at 30 °C,
420 rpm agitation, and operating pressure with 59 Lpm air flow). Theoretical evaporative losses were estimated at 0.12 kg/h
for the 100-L vessel and actual water loss was measured at 0.06 kg/h after a 16 h hold step (see Table III).
Since the 15-L vessel used an exhaust condenser during normal operation, water losses would be negligible; however, for comparison,
a theoretical evaporation rate of 0.019 kg/hr was calculated for the 15-L fermenter (10 Lpm air flow at 30 °C and atmospheric
pressure). Actual water loss without the use of the exhaust condenser was measured at 0.0096 kg/h after a 44 h hold step (11
L water at 30 °C, 800 rpm agitation, and operating pressure with 10 Lpm air flow).
Transfer of the adapted process
Based on the equipment characterization results, it was concluded that the pilot scale 100-L fermentation system would be
suitable for high density cultivation as long as the oxygen transfer requirements could be met. Since the kLa measured in water for the pilot system was approximately 86% of that for the laboratory scale system, it was assumed that
the peak OUR supported by this system would also be lower. Thus, it was decided to decrease the duration of the exponential
feed phase to 7 h to lower oxygen requirements. Based on the laboratory scale OUR data, this would decrease the peak oxygen
uptake rate to about 80% of that observed in the 15-L fermenter. Again, the induction medium would be added at a constant
rate equal to the feed rate reached at the end of the exponential feed phase. Another change was to set the exponential feed
start time to 7 h after inoculation. At the laboratory scale, the feed start time varied between 7 and 8 h based on a rapid
increase in DO that indicated the complete consumption of glycerol in the batch medium.
Figure 10: Growth data at the pilot scale. Dotted line shows a typical lab scale fermentation. Solid lines show two batches
completed at the 100-L pilot scale with reduced feeding time.
Q-beta VLP fermentation runs were successfully executed in the pilot plant after further adaptation of the laboratory-scale
process as described above. Growth performance monitored by OD600 as well as other online parameters were as expected. The final cell density and titer were lower than the corresponding laboratory-scale
values due to a shorter exponential feed stage; however, Q-beta monomer productivity (g/g DCW) was comparable (see Figure
10, Table IV). Given the program requirements for VLP material, the fermentation titer was deemed to be acceptable. Product
quality after purification was tested with several assays, including those for SEC-HPLC, SDS-PAGE, peptide mapping, RNA content
and host cell protein content. Sample SEC-HPLC profiles are shown in Figure 6B. Based on all the analytical results, the
produced VLP was found to be within the desired specifications.
Table IV: Summary of fermentation data.